Stem cuttings from P. dulcis (Trev.) Mold. (Verbenaceae) growing in a peat moss potting medium in plastic containers in a greenhouse were planted on raised beds in March-April, 2013 in the medicinal plants garden of Prairie View A & M University Research Farm, and watered by drip irrigation as necessary without fertilizer treatment. The soil at the site is characterized as a fine-loamy, siliceous, thermic Typic Paleudalf. The weather conditions throughout the Phyla establishment were monitored at the USDA-NRCS Scan Station on the Prairie View A & M University farm a few meters from the Phyla plot. The Phyla established rootings quickly and commenced good vegetative growth. In late April, green and young leaves were harvested from many healthy plant stands, frozen in dry ice and transported to storage in −80˚C freezer. The P. dulcis continued to grow in the plots, and in June-July the intense summer light and heat of Texas induced the purple coloration of the older leaves.

GDH was extracted with Tris-HCl buffer solution containing RNAse A and DNase 1, and purified by electrophoresis as described before [21] [22] from P. dulcis green leaves harvested from the medicinal plants garden. RNA synthetic activity of GDH isoenzymes [12] was assayed in combined deamination and amination substrate solutions of 0.1 M Tris-HCl buffer solution (pH 8.0) containing the four NTPs (0.6 mM each), CaCl₂ (3.5 mM), L-glu (3.23 μM), NAD⁺ (0.375 μM), NH₄Cl (0.875 mM), α-ketoglutarate (10.0 mM), NADH (0.225 mM), 5 Units RNase inhibitor, 1 Unit DNase 1, and 5 μg of actinomycin D. Reaction was started by adding 0.2 mL of whole gel-eluted GDH charge isomers containing 3 - 6 μg protein per mL. Final volume of the reaction was brought to 0.4 mL with 0.1 M Tris-HCl buffer pH 8.0. Reactions were incubated at 16˚C overnight and stopped by phenol-chloroform (pH 5.5) extraction of the enzyme. RNA was precipitated with ethanol, and dissolved in minimum volume of molecular biology quality water. RNA yield and quality were determined by photometry and by agarose gel electrophoresis. Assays were carried out in duplicate to verify the reproducibility of the results.

cDNAs were synthesized with 2 μg of each product RNA synthesized by the whole gel-eluted GDH charge isomers using random hexamer primer. Restriction fragment PCR amplification; adapter ligation; sequencing gel fractionation; and purification of cDNA fragments [12] [13] were conducted according to the methods of Display Systems Biotech, Vista, CA, USA. Selected cDNA fragments were subcloned into pCR4-TOPO vector and transformed into TOP10 One Shot Chemically Competent Escherichia coli (Invitrogen, Carlsbad, CA), followed by overnight growth on selective plates. Up to ten positive transformant colonies were picked per plate and cultured overnight in LB medium containing 50 μg/mL of kanamycin. Plasmid DNA was purified with a plasmid kit (QiaGen mini kit, Madison, WI). The insert cDNA was sequenced with T3 and T7 primers by Functional Biosciences, Inc. (Madison, WI, USA).

To characterize the GDH-synthesized RNAs, the cDNA sequences were used as queries to search the NCBI nucleotide-nucleotide (excluding ESTs) BLAST (blastn), and non-redundant protein translation (blastx) databases for Phyla dulcis and related taxids. Complementary DNAs that displayed the highest alignment scores with mRNAs encoding the enzymes of primary and natural products (secondary) metabolism were selected.

Phyla dulcis, prostate perennial medicinal herb with many branches, originally a South American sweet herb has been established in the medicinal plants garden of the University, Waller County, Texas, USA. The weather conditions for March and April 2013 (Table 1) showed that rainfall decreased but temperatures increased during the period of the experiment. The weather conditions are important for reporting experimental conditions and for future field plot establishment of the species. Under the intensive light, heat and humidity conditions of Texas summer, the older leaves turned purple (Figure 1) suggesting that the photosynthetic pathways could be subject to environmental regulation. There is no literature record on the sustainable cultivation of the species in the USA, therefore environmental research is in progress for identifying best management strategies (fertilization, plant protection, irrigation etc.).

Rotofor isoelectric focusing (IEF) of P. dulcis GDH extracts gave a single horizontal row of isoenzymes covering from Rotofor chambers (fractions) 4 to 12 on native PAGE (figures not shown) unlike that of peanut [22] . RNA synthetic assays of the whole-gel eluted isoenzymes gave a series of predominantly low molecular weight

^†The weather data were obtained from the USDA-NRCS Scan Station on the Prairie View A & M University farm a few meters from the Phyla plot; ^‡Monthly mean and standard deviation.

(<500 nucleotides long) RNAs on agarose gels (figures not shown) irrespective of the pI values of the isoenzymes. Therefore, the acidic isoenzymes (Rotofor fractions 4 to 6) were combined, neutral isoenzymes (Rotofor fractions 7 and 8) were combined, and basic isoenzymes (Rotofor fractions 9 to 12) were combined and the three composite samples were applied for the double differential display analyses.

The purpose of the differential display RF-PCR of the GDH-synthesized RNAs was to permit easy molecular biology amplification, isolation, and bioinformatics characterization of the cDNA fragments homologous to P. dulcis mRNAs as were done previously [21] . The double differential display patterns of the cDNAs (Figures 2-4) showed that the RNAs synthesized by the acidic, neutral, and basic isoenzymes of Phyla GDH were structurally different isomers in agreement with the binomial subunit compositions in the hexameric isoenzymes [21] -[25] . All the 64 display PROBES of Display Systems Biotech, Vista, CA, USA were studied. Based on the subsets of cDNA fragments obtained, the PCR priming by display PROBES 1, 4, and 18 (Figures 2-4) showed that the neutral isoenzymes of GDH were more active in RNA synthesis than the acidic and basic isoenzymes. The display patterns obtained in replicate RF-PCR experiments showed complete identity per display PROBE. Similarly, the differential band patterns for GDH-synthesized RNAs were different from those for mRNAs [26] . Therefore the synthesis of RNA by P. dulcis GDH was specific and reproducible reactions. cDNAs of GDH- synthesized RNAs are important Northern hybridization tools for monitoring and quantitation of the abundances of mRNAs that are homologous to them [12] -[17] .

To assign functions to the cDNA fragments, their sequences were used to search the GenBank databases with the BLASTN etc. algorithms. An overview of the mRNA targets homologous to the differential cDNA fragments (Figures 2-4) showed that overwhelmingly the mRNAs encoding the enzymes of primary metabolism were the targets of the RNAs synthesized by the acidic GDH isoenzymes; whereas predominantly the mRNAs encoding the enzymes of natural products metabolism were the targets of the RNAs synthesized by the neutral and basic GDH isoenzymes. These are important molecular differences and similarities that would allow environmental conditions to be focused either on the primary or terpenoid metabolism of the plant species.

Differential cDNA band 1.02 (Figure 2) yielded several isomeric cDNAs (Table 2). They showed sequence homologies with mRNAs encoding Phyla primary metabolic enzymes including dehydroquinate dehydratase, small subunit of ribulose-1,5-bisphophatase carboxylase, cytochrome P-450 reductase, granule-bound starch synthase, pyruvate kinase, glucose-6-phosphate dehydrogenase, protoporphyrinogen oxidase, phosphoenol pyruvate carboxylase, quinolinate phosphirobosyltransferase, photosystem II protein, and coumarate: coenzyme A ligase. The mechanism of GDH-synthesized RNA is that they silence mRNAs homologous to them [16] [24] . All the mRNAs homologous to cDNA fragments 1.02 (Figure 2) have different molecular weights therefore making them very easy to identify on Northern blot analyses. This is a considerable advantage in target mRNA profiling and modeling of biochemical pathways [14] [17] . Differential cDNA fragment 1.03 (Figure 2) was homologous to mRNA encoding peroxidase. The metabolic probes embodied by the cDNA fragments 1.02 and

1.03 constitute important molecular tools for monitoring and regulating P. dulcis mRNA targets in photosynthesis (Figure 1). The dual importance of GDH-synthesized RNAs is that in vivo they silence mRNAs homologous to them; and in vitro they serve as reliable probes in Northern hybridization analyses [12] -[16] .

Complementary DNA fragments 4.20 (Figure 3) showed sequence homologies with mRNAs encoding Phyla terpenoid metabolic enzymes including β-caryophyllene synthase, (+)-epi-α-bisabolol synthase, bicyclogermacrene synthase, α-copaene/δ-cadinene synthases, trans-α-bergamotene synthase, terpene synthase 1, bifunctional sesquiterpene synthase, and geraniol synthase (Table 2). These are some of the terpene synthases that have been characterized before [7] [11] ; and the encoding mRNAs are related to the enzymes that synthesize some of the undesirable secondary metabolites of Phyla [4] [10] . Complementary DNA fragments 4.22 (Figure 3) similar to fragments 4.20 showed sequence homologies with mRNAs encoding Phyla terpenoid metabolic enzymes including in addition prenyltransferase (Table 2). All the mRNA homologous to cDNA fragments 4.20 and 4.22 possess different molecular weights therefore making them very easy to profile on Northern blot analyses. The metabolic probes embodied by cDNA fragments 4.20 and 4.22 constitute important molecular tools for monitoring and regulating Phyla biochemical targets in the study of the effects of agronomic management factors (fertilization, irrigation, pesticide tolerance) on secondary metabolism.

RNAs synthesized by the basic isoenzymes of Phyla GDH (lane C-18 of Figure 4) gave differential cDNA fragmentation patterns similar to those of the RNAs synthesized by the neutral isoenzymes (lanes B-1, B-4, B-18 of Figure 4). Complementary DNA fragments 18.16 (Figure 4) shared sequence homologies with mRNAs encoding β-caryophyllene synthase, α-copaene/δ-cadinene synthases, and trans-α-bergamotene synthase, bicyclogermacrene synthase (Table 2) all of which are molecular targets in natural products metabolism. Complementary DNA fragments 18.18 (Figure 4) shared sequence homology with mRNAs encoding β-caryophyllene synthase (Table 2) therefore it will complement fragments 18.16 in Northern profiling of Phyla total RNAs. RNA differential display (Figures 2-4) is a sensitive, reproducible, and versatile technology for profiling different RNAs simultaneously [26] .

The GDH-synthesized RNAs that were homologous to mRNAs encoding Phyla primary metabolic enzymes displayed extensive sequence similarities. The cDNA fragment (Table 2) homologous to the mRNA encoding granule-bound starch synthase shared 8-fold plus/minus sequence matches with the cDNA fragment (Table 2) homologous to the mRNA encoding glucose-6-phosphate dehydrogenase; 5-fold plus/minus sequence matches with the cDNA fragment (Table 2) homologous to the mRNA encoding protoporphyrinogen oxidase; but 4-fold plus/plus sequence matches with the cDNA fragment (Table 2) homologous to the mRNA encoding 4-couma- rate: coenzyme A ligase. The RNAs synthesized by GDH are isomeric in structure similar to the binomial subunit structure of the enzyme’s hexamers [25] . Structural similarities between a GDH-synthesized RNA and several mRNAs in different biochemical pathways constitute the channels of cross-talks that differentially regulate or synchronize metabolism [12] -[16] through coordinate silencing as distinct from coordinate expression of transcripts. GDH is present in the cytoplasm, chloroplast, and mitochondria [27] thereby enabling the RNA it synthesizes to regulate different metabolic pathways.

There were also structural similarities between the RNAs synthesized by the acidic, neutral, and basic isoenzymes of P. dulcis GDH. The cDNA fragment (Figure 2) homologous to the mRNA encoding dihydroquinate dehydratase (Table 2) shared 3-fold matches two of which were plus/minus and the remaining one was plus/plus sequence matches with the cDNA fragment (Table 2) homologous to the mRNA encoding β-cryophyllene synthase, a natural products metabolic enzyme. The cDNA fragment (Table 2) homologous to the mRNA encoding granule-bound starch synthase (primary metabolism) shared 5-fold plus/plus sequence matches with the cDNA fragment (Table 2) homologous to the mRNA encoding bifunctional sesquiterpene synthase, a natural products metabolic enzyme. The geographic positioning of the cDNA fragments on the sequencing gel (Figures 2-4) whereby those resulting from the cathodal GDH isoenzymes (north side of the gel) regulated those from the anodal isoenzymes (south side) and vice versa; and those from the acidic GDH isoenzymes (west side) regulated those from the neutral and basic isoenzymes (east side) and vice versa describe the fidelity of the GDH-synthe- sized RNAs in the silencing of mRNAs in vivo, and the fool-proof characteristics of the cDNA fragments as Northern probes. The plus and minus strands of a cDNA fragment as two-probes-in-one potentially assure that the target mRNA will not be missed during Northern hybridization reaction. Furthermore, the multiple internal sequence repeats in the GDH-synthesized RNA assure tenacious crisscross binding of the cDNA fragment to the homologous mRNA thereby maximizing specificity and stringency through liquefaction reaction during Northern hybridization. Single-stranded nucleic acid probes lack the crisscross tenacious binding specificity and stringency embodied by GDH-synthesized RNAs. Geographic positioning on the sequencing gel landscape of the RNAs synthesized by GDH (Figures 2-4) would permit the biochemical selection of specific metabolic chemotypes of P. dulcis as was in the case of peanut [12] -[17] . Therefore, photosynthetic processes regulate secondary metabolism, suggesting that environmental conditions would be able to differentiate the metabolism of terpenoids to produce several Phyla chemotypes. Aside from the general fact that the initial substrates for secondary metabolism are sourced from primary metabolism, the cross-connections at the mRNA level (coordinated silencing) between primary and secondary metabolic pathways could potentially provide a practical approach for molecular regulation of terpenoid accumulation. The superimposition of a rainbow of environmental conditions on Phyla growth and development may manipulate the inter-mRNA cross-talks to the extent that special metabolic chemotypes arise some of which could be high hernundulcin thus illuminating possible alternative chemical treatment approaches [5] [6] for the cultivation of the medicinal plants.

By knocking down biomass and fatty acid metabolic pathways, GDH-synthesized RNAs enhanced the production of arachin-free low linoleic acid [14] , and ultra-high resveratrol [17] peanut kernels. Similar technologies could be readily patterned for knocking down aspects of Phyla photosynthetic or monoterpene metabolism in order to enhance sesquiterpene accumulation. Several poor or disaster-afflicted rural communities exist worldwide [28] . Governments and humanitarian agencies are reaching out to alleviate the sociological and agricultural stresses of those communities. Phyla dulcis now grows luxuriantly in USA (Figure 1). Phyla species are money-makers [2] for small-scale farmers promoting rural economy in South Africa, India, China, and South America. High hernundulcin metabolic variants of the medicinal plants could enable limited resource farmers to increase their farm income thereby promoting rural economy world-wide besides complementing the industrial production of hernundulcin [1] [8] [9] . One of the major predispositions to obesity is the consumption of high calorie food. Sugar is one of the high calorie ingredients added in to food. The zero-calorie hernundulcins could possess the potential to minimize sugar intake while offering sweet taste to sweet-consumers. High-hernundul- cin Phyla-based natural sweetener, similar to stevia could be popular in the health-conscious market place.